1、Designation: D 4412 84 (Reapproved 2002)Standard Test Methods forSulfate-Reducing Bacteria in Water and Water-FormedDeposits1This standard is issued under the fixed designation D 4412; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision
2、, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 These test methods cover the procedure for the detectionand enumeration by the most probable number (MP
3、N) tech-nique of sulfate-reducing bacteria in water or water-formeddeposits.1.2 Two media preparations are provided. Medium A whichis prepared with reagent grade water, and Medium B which isprepared using the water to be sampled as the water source.Medium B is offered for those special conditions wh
4、eresulfate-reducing bacterial strains have adapted to atypicalnon-fresh water environment.1.3 For the isolation and enumeration of thermophilicsulfate-reducing bacteria encountered in waters associated withoil and gas production, all broths, dilution blanks, and incuba-tions must be maintained at te
5、mperatures of at least 45C andpreferably within 5C at the sample temperature.1.4 The sensitivity of these test methods can be increased bypurging the dilution blanks and tubes of media with nitrogenimmediately prior to use.1.5 The analyst should be aware that adequate collaborativedata for precision
6、 and bias statements as required by PracticeD 2777 are not provided. See Section 11 for details.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health p
7、ractices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:D 1129 Terminology Relating to Water2D 1193 Specification for Reagent Water2D 2777 Practice for Determination of Precision and Bias ofApplicable Methods of Committee D19 on Wate
8、r2D 3370 Practices for Sampling Water from Closed Con-duits22.2 APHA Standard:Standard Methods for the Examination of Water and Waste-water, Fifteenth Edition33. Terminology3.1 DefinitionsFor definitions of terms used in these testmethods, refer to Terminology D 1129.3.2 Definitions of Terms Specifi
9、c to This Standard: For adescription of the term MPN used in these test methods, referto literature.44. Summary of Test Methods4.1 Water and water deposit samples and dilutions of thesesamples are dispensed into tubes of Starkeys medium (Aor B)following five tube MPN procedures. The tubes are sealed
10、 withliquid paraffin, and incubated at 20C for 21 days.4Positivereactions are indicated by the deposit of a black precipitate.5. Significance and Use5.1 Sulfate-reducing bacteria are widely distributed in ma-rine and fresh water muds which, in consequence, frequentlyare laden with the hydrogen sulfi
11、de produced by these organ-isms during dissimilatory sulfate reduction.5.2 It has been reported that Desulfovibrio can form asmuch as 10 g of sulfide per litre during active multiplication.Sulfate-reducing bacteria can cause the external or internalcorrosion of water or wastewater pipelines and pipe
12、lines forpetroleum and natural gas. The formation of galvanic cells bymassive growth of sulfate-reducing bacteria under suitableconditions makes the corrosion much worse than just the effectof the hydrogen sulfide on the metal or concrete.6. Apparatus and Materials6.1 Anaerobic Incubator, 20C, if av
13、ailable, or conventional20C incubator.56.2 Pipets, sterile, 1 mL and 10 mL, “calibrated” to deliver.6.3 Test Tubes, with close fitting or airtight caps; 16 by 150mm and 20 by 150 mm.1These test methods are under the jurisdiction of ASTM Committee D19 onWater and are the direct responsibility of Subc
14、ommittee D19.24 on Water Micro-biology.Current edition approved Oct. 26, 1984. Published February 1985.2Annual Book of ASTM Standards, Vol 11.01.3Available from American Public Health Association, 1015 18th St. N.W.,Washington, DC 20036.4Bonde, G. J., “Bacterial Indicators of Water Pollution,” A Stu
15、dy of QuantitativeEstimation, Teknisk Forlag, Copenhagen, 1963.5For thermophilic organisms use a 45C incubator.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6.4 Test Tube Racks, of sufficient size to contain 16 and20-mm tubes.7. Re
16、agents7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society,6whensuch specifications are available.7.2 Purity of WaterU
17、nless otherwise indicated, referencesto water shall be understood to mean Reagent Water Type IIconforming to Specification D 1193. In addition, reagent waterused for these test methods must be sterile.7.3 Starkeys Medium A7(modified):Sodium lactate (C3H5NaO3) 3.5 gAmmonium chloride (NH4Cl) 1.0 gDipo
18、tassium, hydrogen orthophosphate(K2HPO4)0.5 gMagnesium sulfate (MgSO47H2O) 2.0 gSodium sulfate (Na2SO4) 0.5 gCalcium chloride (CaCl22H2O) 0.1 gThioglycollic acid 0.1 gAmmonium ferrous sulfate or ferrousammonium sulfate(NH4)2SO4FeSO46H2O)0.001gWater (H2O) 1 L7.3.1 Double strength medium (23) is prepa
19、red as aboveexcept 500 mL of water are used instead of 1 L.7.3.2 Heat to dissolve and dispense 9 mL of medium persingle strength tube, and 10 mL per double strength tube.7.3.3 Tubes should be of sufficient capacity to contain 1 mLof inoculum plus 9 mL of single strength medium or 10 mL ofinoculum pl
20、us 10 mL of 23 medium.7.3.4 pH of medium should be 7.2 after autoclave steriliza-tion, at 121C for 15 min.7.4 Starkeys Medium BThe medium is similar to thatdescribed in 7.3, 7.3.1, and 7.3.2 with the following modifica-tion:7.4.1 Water collected from the sample collection site is usedto prepare the
21、medium outlined in 7.3. The water sample isfiltered to remove particulates (1.2 m membrane filter) and thepH is recorded.7.4.1.1 After preparing the Medium B following 7.3.1,7.3.2, and 7.3.3, and prior to dispensing, check and adjust pH,if necessary to that of the original water used, then filterste
22、rilize the medium by passage through 0.2-m filter andasceptically dispense into presterilized tubes.7.5 Hydrogen Sulfide Test Reagent:7.5.1 Ferric Chloride Stock Solution (FeCl36H2O)Dissolve 13.5 g of ferric chloride in a mixture of 250 mL ofwater and 250 mL of HCl (sp gr 1.19). Store in an airtight
23、amber container. Prepare fresh monthly.7.5.2 p-Aminodimethylaniline Dihydrochloride Stock Solu-tionp-Aminodimethylaniline dihydrochloride(C8H12N22HCl)1.0 gHCl (6 N) 500 mLDissolve1gofp-aminodimethylaniline dihydrochloride in500 mL of 6 N HCl. Store for up to 1 month in an amberairtight container.7.6
24、 Liquid ParaffnHeavy, sterile, or sterile mineral oil.7.7 Buffered Dilution WaterStock Solution7.7.1 Dissolve 34.0 g of KH2PO4in 500 mL of water, adjustpH to 7.2 with 1 N NaOH and dilute to 1 L with distilled water.This is called the stock phosphate solution.7.7.2 Dissolve 38 g of MgCL2in 1 L of dis
25、tilled water.7.8 Buffered Dilution Water, Working SolutionAdd 1.25mL of stock buffered dilution water and 5 mL of MgCl2solution to 500 mL of water. Bring to 1 L with water. Mix welland dispense as 90 mL dilution blanks in screw-capped bottles.Sterilize by autoclaving at 121C for 15 min.8. Procedure8
26、.1 Clean and disinfect the area with a cleaning solution thatleaves no residue.8.2 Set out and label five replicate tubes of 10-mL double-strength Starkeys medium, A or B, in the test tube rack.8.3 Set out and label five replicate tubes of 10-mL single-strength Starkeys medium, A or B, for each mL o
27、f sample ormL of sample dilution to be tested. Use two sets of fivereplicate 10-mL tubes, each to contain 1 mL of sample or 1 mLof 1/10 dilution of sample.8.4 Prior to sample inoculation, heat tubes of media anddilution blanks in a water bath to 60C then cool rapidly to20C to ensure minimal oxygen l
28、evels.8.5 Shake sample thoroughly,8at least 25 times; makedilutions starting with 10 mL of sample into one 90-mLdilution blank.8.6 Pipet 10 mL of sample into each double-strength brothand 1 mL of sample or diluted sample into each set of fivesingle-strength broths.8.7 Maintain anaerobic conditions b
29、y layering 2 to 3 ml ofsterile liquid paraffin in each tube.8.8 Recap tubes and incubate at 20C for 21 days.8.9 Include sterile water samples with each test as negativecontrols.8.10 Positive reaction is indicated by the deposit of a blackprecipitate (sulfide).8.11 Confirm dubious results by the addi
30、tion of 0.5 mL offerric chloride reagent followed by 0.5 mL ofp-aminodimethylaniline reagent to the MPN tube. Add reagentto the bottom of the tube using syringe or long Pasteur pipet. Apositive reaction, blue color, occurs within 10 min if H2Sispresent.6Reagent Chemicals, American Chemical Society S
31、pecifications, AmericanChemical Society, Washington, DC. For suggestions on the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Co
32、nvention, Inc. (USPC), Rockville,MD.7Starkey, R. L., “Characteristics and Cultivation of Sulfate-Reducing Bacteria,”Journal of the American Water Works Association, Vol 40, 1948, pp. 12911298.8Organisms do not appear to be hypersensitive to small amounts of oxygen.D 4412 84 (2002)29. Calculation9.1
33、Compute the number of positive findings resulting frommultiple-portion decimal dilution planting as the combinationof positives and recorded in terms of the Most ProbableNumber3(MPN).9.2 When more than three series of tubes are employed in adecimal series of dilutions, use the results from only thre
34、e ofthese used in computing the MPN, for example:10mL1mL0.1mL0.01mL5/5 5/5 2/5 0/5 = 5-2-0 3 10 = 490/100 mL5/5 4/5 2/5 0/5 = 5-4-2 = 220/100 mL5/5 3/5 1/5 1/5 = 5-3-2 = 140/100 mL5/5 0/5 0/5 0/5 = 5-0-0 = 23/100 mL10. Report10.1 Report the results as number of sulfate-reducing bac-teria per 100 mL
35、of sample.11. Precision and Bias911.1 Due to the instability of the organisms, round robintesting can not be carried out. Statements can only be made onthe precision of the MPN procedure.11.2 Unless a large number of portions of sample areexamined, the precision of the MPN is rather low. For example
36、,even when the sample contains one organism per millilitre,about 37 % of tubes inoculated with 1 mL of sample may beexpected to yield negative results because of irregular distri-bution of the bacteria in the sample and the multiple attachmentof bacteria to particles. When five tubes, each with 1 mL
37、 ofsample, are employed under these conditions, a completelynegative result may be expected less than 1 % of the time.Thus, even when five tubes are employed for each dilution, theprecision of the results obtained is not of a high order.11.3 See the Research Report for results of a single labora-tor
38、y, two operator study.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringeme
39、nt of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or
40、for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views kno
41、wn to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).9Supporting data for these test methods have been filed at ASTM Headquarters.Request RR: D19-1116.D 4412 84 (2002)3
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